Primary Research On MiRNAs In Malpighian Tubule From The Silkworm Larvae(Bombyx Mori) Response To BmNPV Infection

Bombyx mori is a model insect of Lepidoptera, and the innate immune defensemechanism is a hot research topic recently. Malpighian tubule of insect is an autonomousimmunologic tissue, which can start an effective immune response entirely independent ofthe fat body. Our research group has been confirmed that Malpighian tubules of silkwormhave immune regulatory function at the protein level. Recent studies show that miRNAs inorganisms play an important role in anti-viral infection. In this paper, we explore researchon Malpighian tubules of silkworm response to BmNPV virus infection at miRNA levelsand obtained the preliminary results as following.1. Small RNA library construction of Malpighian tubules and deep sequencingOn the1stday of fifth instar, silkworm larvae were injected with BmNPV-EGFP andTC-100. The Malpighian tubules tissues were collected at3h,6h,12h and24h posttreatment, respectively. By using Illumina Solexa high-throughput sequencing platform, twopooled small RNA libraries of CK and NPV were constructed and deep sequenced. Theamount of original small RNA sequences in the CK library and NPV library were16,681,601and16,509,994, respectively. The bioinformatics analysis results showed thateffective data retention of2libraries is high as76.5%. The length of valid data is mainlydistributed around the22bp, which is the typical size range for Dicer-derived product, so itcan be used for further information analysis. By blasting against the GenBank database,rRNA, tRNA, snRNA, snoRNA degradation fragments of the two library data were removed.313kind of known miRNA were detected, which54are common between the CK libraryand NPV library. DEGseq analysis result indicated that there are53kind of differentiallyexpressed miRNAs. By blasting against the silkworm genome sequence,108kind of novelmiRNAs were predicted in CK and NPV library. According to differences of sequence copynumber between2libraries,28kind of miRNA, including11known miRNA and17novelmiRNA, were selected as candidate for the further experiment.2. Experimental verification of candidate miRNAs encoded by Bombyx moriMalpighian tubules response to BmNPV infectionThe stem-loop RT-PCR and sequence experiments results showed that11kind ofmiRNAs were identified as correct among28kind of candidate miRNA, including4kind of known miRNAs （bmo_miR₃75, bmo_miR₂731, bmo_miR₉27, bmo_miR₇） and7kindof novel miRNAs （miR_N11, miR_N13、miR_N33, miR_N49, miR_N74, miR_N80,miR_N89）. Then, the relative expression level of11kind of miRNAs in Malpighian tubulestissues between BmNPV treatment and the control was investigated by RT-PCR method.The results showed that expression level of miRN49and miRN74in the control weresignificantly higher than the BmNPV treatment, while bmo-miR₃75had significantlyhigher expression level in the BmNPV treatment than in the control. The relative expressionlevel of other8kind of miRNAs presented no significant difference. So we selected thebmo-miR₃75as the candidate miRNA for the further expression analysis and targetpredictions.3. Expression level of bmo-miR₃75at different infection period of BmNPV andits target gene predictionOn the1stday of fifth instar, silkworm larvae were injected with BmNPV-EGFP andTC-100. The Malpighian tubules tissues were collected at3h,6h,12h,24h,48h,72h,96hand120h post treatment, respectively. The semi-quantitative RT-PCR results showed thatbmo-miR₃75was expressed in each infection period of BmNPV and in the control. Exceptfor48h of infection period, the expression level of bmo-miR₃75is higher in the BmNPVtreatment than that of control, especially in12h,24h and96h. After BmNPV infection, therelative expression levels of bmo-miR₃75was increased gradually at6h,12h and24h, thendecreased slightly at48h and72h, increased sharply at96h and decreased at120h.For theincreased expression trend of bmo-miR₃75at12h,24h and96h in viral infection treatment,we speculated that the silkworm may regulate replication process-related gene expression ofBmNPV in Malpighian tubules by encoding bmo-miR₃75. For the decreased expressiontrend of bmo-miR₃75at48h,72h and120h, we deduced that it may be related to the virusblock the host miRNAome by modulating some of the key components of the miRNAbiogenesis pathway in its own favor in order to counterattack the miRNA-mediated hostdefense. As for the control group, bmo-miR₃75had higher relative expression level at48hthan that of the other periods, which requires further experiments to elucidate.Target gene of bmo-miR₃75in the silkworm genome and the viral genome waspredicted by bioinformatics software of PITA, RNAhybrid and RNA22. The results showedthat the possible targets of bmo-miR₃75in the silkworm genome are cuticular proteinRR-1motif12, Ultraspiracle2, myb, and30K lipoprotein precursor. The possible targets ofbmo-miR₃75in the BmNPV genome are DUF1477, P47, ChtBD2, and Baculo_Y142. P47 is viral transcription factor-RNA polymerase subunits involved in the process of viralreplication, which suggested that the Malpighian tubules maybe inhibit directly or indirectlythe BmNPV proliferation by encoding bmo-miR₃75.The expression profile of bmo-miR₃75in cuticle, fatbody, posterior silk gland,trachea, testis, blood and malpighian were investigated. The results showed thatbmo-miR₃75could not be detected in the midgut and blood in the control. However, it wasexisted in all tissues of the BmNPV treatment. More interesting, expression level ofbmo-miR₃75in the posterior silk gland is relatively high in both treatments. Compared tothat of the control, the expression level of bmo-miR₃75in the posterior silk gland ishighest in the BmNPV treatment. The above results inferred that bmo-miR₃75maybeplays an important biological role in the posterior silk gland.The current research results may provide some basic information about immuneregulation mechanism between Malpighian tubules of silkworm and the virus at miRNAlevels. What mechanism does miRNA participate in the immune regulation needs furtherexperiment to elucidate in the future.